1. Field of the Invention
The present invention relates generally to the use of molecular biological techniques in the cloning, identification and characterization of a mammalian diphtheria toxin (DT) receptor. In particular, the invention relates to DNA segments and recombinant vectors encoding a DT receptor, to the expression of the biologically active DT receptor in recombinant cells, to methods for conferring DT sensitivity to a cell, and to methods for the identification of candidate substances that bind to the DT receptor. It is also envisioned that the DT receptor may be of use in the development of novel drug delivery systems, and as a negative-selectable marker in gene transfer systems.
2. Description of the Related Art
Diphtheria toxin (DT), a potent exotoxin produced by lysogenized strains of Corynebacterium diphtheriae, is a multifunctional protein that kills susceptible mammalian cells. It is composed of two disulfide-linked protein fragments, both of which are required for the intoxication process (Collier, 1975; Pappenheimer, 1977; Eidels et al., 1983; Middlebrook & Dorland, 1984). The A-fragment catalyzes the ADP-ribosylation of eukaryotic elongation factor 2, thereby inhibiting protein synthesis. The B-fragment is responsible for binding of the toxin to cells and is essential for facilitating the entry of the A-fragment into the cytosol (Collier, 1975; Pappenheimer, 1977; Eidels et al., 1983; Middlebrook & Dorland, 1984).
The existence of specific cell-surface DT receptors was first demonstrated by Ittelson & Gill (1973) employing a competitive inhibition of binding approach, and it is now known that DT enters susceptible mammalian cells via receptor-mediated endocytosis (Morris et al., 1985; Keen et al., 1982). The initial step involves the binding of DT to a specific cell-surface receptor, followed by internalization of the toxin:receptor complexes into coated pits and translocation of the A-fragment into the cytosol. Not all mammalian cells are equally sensitive to DT (Middlebrook & Dorland, 1977b; Middlebrook et al., 1978). For example, monkey kidney cells such as Vero cells, are highly sensitive, whereas human and hamster cells are moderately sensitive and mouse and rat cells are resistant.
As elongation factor 2 of all mammalian cells can be ADP-ribosylated by the DT A-fragment, it is generally believed that the difference in sensitivity to DT between species is due to the number of functional cell-surface DT receptors. In this regard, Vero cells display 1-2.times.10.sup.5 receptors per cell (Middlebrook et al., 1978), whereas mouse and rat cells lack detectable receptors (Eidels et al., 1983; Stenmark et al., 1988). Prior to the present invention, a specific DT receptor has not been completely isolated or characterized, although a 27 kD DT receptor-associated Vero cell protein has recently been reported (Iwamoto et al., 1991).
The present inventors recently isolated and characterized a diphtheria toxin-sensitive (DT.sup.S) mouse cell line obtained by transfection of mouse L-M cells with monkey cell genomic DNA. A replica plate screening procedure was developed and employed to detect and isolate mouse cells that had acquired DT sensitivity (Naglich & Eidels, 1990). The degree of toxin sensitivity of the DT.sup.S mouse cells in this study was only moderate and no specific binding of radioiodinated DT to the cell surface was detected. Although the mode of action of DT after it enters a cell is generally known, prior to the studies disclosed herein, there existed a marked lack of knowledge concerning the DT receptor molecule.